Bottom Line:
GbML1 overexpression in Arabidopsis increased the number of trichomes on stems and leaves and increased the accumulation of anthocyanin in leaves.Taken together, the L1 box binding protein, GbML1 was identified as the first partner for GbMYB25 and the role of START domain was discovered to be a protein binding domain in plants.Our findings will help the improvement of cotton fibre production and the understanding of the key role of HD-Zip family and MYB family in plants.

ABSTRACTTranscription factors play key roles in plant development through their interaction with cis-elements and/or other transcription factors. A HD-Zip IV family transcription factor, Gossypium barbadense Meristem Layer 1 (GbML1) has been identified and characterized here. GbML1 specifically bound to the L1 box and the promoters of GbML1 and GbRDL1. GbML1 physically interacted with a key regulator of cotton fibre development, GbMYB25. Truncated and point mutation assays indicated the START-SAD domain was required for the binding to the C terminal domain (CTD) of GbMYB25. GbML1 overexpression in Arabidopsis increased the number of trichomes on stems and leaves and increased the accumulation of anthocyanin in leaves. Taken together, the L1 box binding protein, GbML1 was identified as the first partner for GbMYB25 and the role of START domain was discovered to be a protein binding domain in plants. Our findings will help the improvement of cotton fibre production and the understanding of the key role of HD-Zip family and MYB family in plants.

Mentions:
Previous studies demonstrated that recombinant ATML1 and PDF2 proteins could bind to the L1 box in vitro (Abe et al., 2001, 2003). EMSA assays were performed by using purified full-length or truncated GbML1 proteins (see Supplementary Fig. S2A at JXB online). Complex formation was observed with the GbML1 (Fig. 2A, lane 5; Fig. 2B) and HD-ZLZ proteins (Fig. 2C, lane 4) but not with the MBP (Fig. 2A, lane 4) or the HD (Fig. 2C, lane 3), START–SAD proteins (Fig. 2C, lane 5). No complex formation was observed with the mutated L1 box (Fig. 2A, lane 6). The third helix of the HD domain was shown to contact the double-stranded DNA, and was important for protein–DNA interaction. Two mutations were made in this region, L94P and N107I (Fig. 2D, left; see Supplementary Fig. S2B at JXB online). While the L94P mutation greatly reduced the interaction between the HD-ZLZ protein and the L1 box, the N107I mutation abolished the interaction (Fig. 2D, right). Moreover, GbML1 could bind to the L1 box containing parts from GaRDL1 (Li et al., 2002) and GbML1 promoters in vitro (Fig. 2E; see Supplementary Fig. S3 at JXB online). These results indicate that GbML1 can specifically bind to L1 box and may specify the gene expression in the L1 layer.

Mentions:
Previous studies demonstrated that recombinant ATML1 and PDF2 proteins could bind to the L1 box in vitro (Abe et al., 2001, 2003). EMSA assays were performed by using purified full-length or truncated GbML1 proteins (see Supplementary Fig. S2A at JXB online). Complex formation was observed with the GbML1 (Fig. 2A, lane 5; Fig. 2B) and HD-ZLZ proteins (Fig. 2C, lane 4) but not with the MBP (Fig. 2A, lane 4) or the HD (Fig. 2C, lane 3), START–SAD proteins (Fig. 2C, lane 5). No complex formation was observed with the mutated L1 box (Fig. 2A, lane 6). The third helix of the HD domain was shown to contact the double-stranded DNA, and was important for protein–DNA interaction. Two mutations were made in this region, L94P and N107I (Fig. 2D, left; see Supplementary Fig. S2B at JXB online). While the L94P mutation greatly reduced the interaction between the HD-ZLZ protein and the L1 box, the N107I mutation abolished the interaction (Fig. 2D, right). Moreover, GbML1 could bind to the L1 box containing parts from GaRDL1 (Li et al., 2002) and GbML1 promoters in vitro (Fig. 2E; see Supplementary Fig. S3 at JXB online). These results indicate that GbML1 can specifically bind to L1 box and may specify the gene expression in the L1 layer.

Bottom Line:
GbML1 overexpression in Arabidopsis increased the number of trichomes on stems and leaves and increased the accumulation of anthocyanin in leaves.Taken together, the L1 box binding protein, GbML1 was identified as the first partner for GbMYB25 and the role of START domain was discovered to be a protein binding domain in plants.Our findings will help the improvement of cotton fibre production and the understanding of the key role of HD-Zip family and MYB family in plants.

ABSTRACTTranscription factors play key roles in plant development through their interaction with cis-elements and/or other transcription factors. A HD-Zip IV family transcription factor, Gossypium barbadense Meristem Layer 1 (GbML1) has been identified and characterized here. GbML1 specifically bound to the L1 box and the promoters of GbML1 and GbRDL1. GbML1 physically interacted with a key regulator of cotton fibre development, GbMYB25. Truncated and point mutation assays indicated the START-SAD domain was required for the binding to the C terminal domain (CTD) of GbMYB25. GbML1 overexpression in Arabidopsis increased the number of trichomes on stems and leaves and increased the accumulation of anthocyanin in leaves. Taken together, the L1 box binding protein, GbML1 was identified as the first partner for GbMYB25 and the role of START domain was discovered to be a protein binding domain in plants. Our findings will help the improvement of cotton fibre production and the understanding of the key role of HD-Zip family and MYB family in plants.